Treatment of HIV-infected patients with antiretroviral therapy (ART) has effectively suppressed viral replication; however, the central nervous system (CNS) is still a major target and reservoir of the virus leading to the development of HIV-1-associated neurocognitive disorders (HAND). Importantly, since the beginning of the HIV epidemic, drug use has remained a primary risk factor for contracting, transmitting, and worsening the outcomes of HIV. In fact, regarding pathogenesis, psychostimulants can induce higher viral loads, reduce CD4 counts, and increase rates of ART resistance. Furthermore, neuropsychological decline is greater in individuals that are cocaine users and HIV-seropositive. Extracellular microvesicles (EVs), which includes microvesicles (MVs) and exosomes, have emerged as a novel biological phenomenon, released by virtually every cell type in the body. We have shown that brain microvasculature endothelial cell (BMVEC)-derived EVs contain tight junction proteins (TJPs) and transporter proteins, which are main constituents of the BBB. Furthermore, a hallmark feature of HAND is the disruption of the BBB and loss of TJ complexes. In this proposal, we show that BMVECs shed EVs in response to HIV virotoxins and psychostimulants. Thus, we hypothesize that HIV infection and/or drugs of abuse triggers EV release leading to BBB instability and facilitation of neuroinvasion by infected immune cells. The innovative nature of this proposal is featured in three independent aims. In the first aim, we will characterize the degree of EV production (MVs and exosomes) as a function of psychostimulant type using a novel microfluidic model of the neurovascular unit with primary human cells. We will also investigate the effects of exposure to HIV virotoxins and ART pharmacologic agents on BMVEC-EV production. Furthermore, we will correlate EC-EV release to the phases of addiction in an in-vivo self-administration model as well as in a humanized mouse model of HIV infection with or without drug administration. In the second aim, we introduce the novel concept that BMVEC-derived EVs bind to activated or infected monocytes, which triggers increased monocytic transendothelial migration. Thus, we hypothesize that monocytes utilize TJPs on EVs to engage endothelial tight junction complexes and facilitate immune infiltration of the CNS. In the third aim, we will explore a therapeutic strategy that could minimize EV production and thus rescue BBB integrity. The above will be accomplished by targeted inhibition of ARF6, which is involved in MV and exosome biogenesis. Using new pharmacological tools to inhibit ARF6, we aim to stabilize the BBB to prevent BBB barrier dysfunction during neuroinflammation. The studies proposed herein will offer crucial insight to brain endothelial EV production, mechanisms of immune cell infiltration into the CNS, and also reveal targets for pharmacological interventions that promote BBB protection in HIV and substance use disorder.